Temperature-sensitive mutants of Toxoplasma gondii will be isolated after chemical mutagenesis using a human fibroblast cell. Mechanical transfer of clones established in microwells will be used to simplify the testing of clones for temperature sensitivity. Newly isolated mutants will be examined for specific defects in protein and RNA synthesis in temperature shift experiments. These temperature shift experiments will employ human fibroblast cells labeled with uracil, a technique that allows specific incorporation of the precursor into parasite nucleic acids. We also plan to devise comparable methods for the specific incorporation of radioactive precursors into parasite proteins. Several mutants of Toxoplasma gondii resistant to analogues of nucleosides have been isolated. The biochemical basis of these mutations will be examined by measuring the appropriate kinases in infected and uninfected cells. If necessary the extracts of infected cells will first be resolved by acrylamide gel electrophoresis in order to distinguish cellular from parasite kinases. BIBLIOGRAPHIC REFERENCES: Pfefferkorn, E.R. and L.C. Pfefferkorn, Toxoplasma gondii: Isolation and preliminary characterization of temperature-sensitive mutants. Experimental Parasitology 39. 365 (1976).